CN109811062B - FSHR gene specific SNP marker, detection method of Tian Qiaoda lambing number character of red sheep and application of detection method - Google Patents
FSHR gene specific SNP marker, detection method of Tian Qiaoda lambing number character of red sheep and application of detection method Download PDFInfo
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Abstract
The invention discloses a detection method of FSHR gene specific SNP markers and Tian Qiaoda lambing number traits and application thereof, and relates to the field of molecular breeding. The SNP marker is derived from sheep FSHR genes, is positioned at 75320741 position on chromosome 3, and has G as an original base and A as a variant base; the nucleotide sequence of the SNP marker is SEQ ID NO.1. The lambing number of individuals with GG genotype of the SNP locus is obviously higher than that of the AA genotype. The nucleotide sequence of the primer is SEQ ID NO.2-4. The SNP marker, the primer and the kit are applied to the detection of the characteristics of the Hetian Yang Changao, and the genotype of the SNP marker is detected to determine how the lambing characteristics of the Hetian sheep to be detected; the SNP, the primer, the kit and the detection method can be used for breeding and screening varieties with more lambs.
Description
Technical Field
The invention relates to the technical field of molecular breeding, in particular to a detection method for FSHR gene specific SNP markers and Tian Qiaoda lambing number traits and application thereof.
Background
Sheep is one of the earliest domestic animals domesticated and raised by human beings, provides rich animal products for human beings, and plays a very important role in animal husbandry. The complex topography, wide geographical area and various ecological environments of Xinjiang make it one of the most abundant areas of sheep raising variety resources in our country. The main representative variety formed by the long-term breeding of the local people in Xinjiang, wherein the neutralization Tian Qiaoda red sheep is also an important mutton sheep variety which is mainly distributed in Pishan county in Xinjiang Hetian, has strong stress resistance, strong disease resistance, extremely coarse feeding cultivation resistance in hot environment and strong drought resistance, and is well known for weaving high-quality carpet wool. And Tian Qiaoda red sheep are mostly bred in groups and naturally bred, single-embryo production is carried out in most cases, but the group is located in Pishan county, the breeding environment is hot and severe, the group fertility is high, the development of breasts and nipples is developed, the number of lambs can reach 3-4 lambs on average, the survival rate of the lambs is high, and the red sheep are ideal groups for lamb meat production organization and have research value. And Tian Qiaoda red sheep are taken as a representative excellent variety of Xinjiang, have uniqueness, are required to be effectively protected by local variety resources, and in addition, the excellent meat production characteristics of the red sheep are not reasonably developed and utilized, so that the development of local economy and Xinjiang mutton sheep industry is greatly unfavorable. Therefore, advanced biotechnology is used, modern breeding technology and traditional breeding methods are combined, the Xinjiang dominant animal husbandry is developed, the sheep breeding degree is improved, the special variety of the high-yield mutton sheep is cultivated, and the quality of mutton animal products is improved, so that the development progress of the Xinjiang sheep industry is accelerated. In the rapid development process of mutton sheep industry in China, the key problems are improving the quality of mutton sheep and increasing the number of delivered commercial products, and the key points of the two are optimizing and applying the breeding technology of mutton sheep. The core of the mutton sheep breeding technology is how to exert the expression potential of the mutton sheep breeding characters. Reproductive performance is one of the important economic traits of sheep, where lambing number is an important factor affecting productivity, is genetically controlled, and is an additive-dominant gene mode of action.
At present, genomics becomes one of methods for researching livestock traits, a gene affecting target traits is predicted by utilizing a genomics technology, and a specific mutation site affecting the target traits is searched by utilizing a molecular marker technology, so that the method has practical significance for improving the economic income of herders and comprehensively improving the lambing level of sheep. SNP markers of sheep lambing numbers are less studied at home and abroad, and researches on functional genes of BMP15, BMPR-IB and GDF9 are concentrated; among them, no study on the influence and the Tian Qiaoda red sheep lambing number SNP markers was reported.
Disclosure of Invention
Therefore, the embodiment of the invention provides a FSHR gene specific SNP marker, a detection method of Tian Qiaoda red sheep lambing number character and application thereof.
In order to achieve the above purpose, the present invention mainly provides the following technical solutions:
in one aspect, the embodiment of the invention provides an FSHR gene specific SNP marker, wherein the SNP marker is derived from a Tian Qiaoda red sheep FSHR gene, the SNP marker is positioned at 75320741 position on a Tian Mianyang chromosome 3, an original base is G, and a variant base is A; the nucleotide sequence of the SNP marker is SEQ ID NO.1.
Preferably, the number of lambings of individuals of GG genotype of the SNP locus is significantly higher than that of individuals of AA genotype.
In another aspect, the embodiment of the invention provides a primer for detecting the SNP marker, and the nucleotide sequence of the primer is SEQ ID NO.2-4.
In still another aspect, an embodiment of the present invention provides a kit for detecting the above SNP markers, which includes the above primer.
In yet another aspect, the embodiment of the invention provides an application of the SNP marker, the primer or the kit in breeding sheep in the field.
In yet another aspect, embodiments of the present invention provide a method of detecting and Tian Mianyang lambing count traits, the method comprising: the lambing number character of the sheep is determined by detecting the SNP markers in the genes of the sheep to be detected.
Preferably, the method comprises the steps of:
extracting genome DNA of the sheep to be detected and sheep in the field;
and (3) taking the DNA as an amplification template, taking the primer as an amplification primer, carrying out competitive allele-specific PCR typing, obtaining a PCR typing result of GG type, AG type and AA type, determining all genotypes of SNP markers according to the typing result, and judging the lambing number character of the sheep to be tested according to the genotypes.
Preferably, the genotype of the SNP is that the lambing number of the GG genotype individuals is significantly higher than the lambing number of the AA genotype individuals.
Preferably, the reaction conditions of the PCR: pre-denaturation at 95℃for 10min, denaturation at 95℃for 20s, circulation for 10 times, extension at 61-55℃for 1min, denaturation at 95℃for 20s, circulation for 26 times, and extension at 55℃for 1min;
the reaction system of the PCR:
the nucleotide sequence of the upstream primer 1 is SEQ ID NO.2;
the nucleotide sequence of the upstream primer 2 is SEQ ID NO.3;
the nucleotide sequence of the downstream primer 1 is SEQ ID NO.4.
In still another aspect, the embodiment of the invention provides an application of SNP markers related to lambing number traits of the Hetian sheep in sheep breeding, and individuals with genotype GG in the SNP markers are selected to breed Hetian sheep varieties with more lambing numbers.
Compared with the prior art, the invention has the beneficial effects that:
the invention utilizes a selection signal detection method and a KASP technology method, namely, a gene related to sheep lambing number character is screened out by utilizing a method for simplifying genome, and the mutation of the gene in the sheep flock of Tian Qiaoda is determined by typing the Chr 3:g.75320741 locus of FSHR gene by a KASP typing method; determining the base position of mutation, performing significance test on the lambing numbers of the Tian Qiaoda red sheep and among different genotypes by using biological statistical software SPSS19.0, judging whether the SNP locus significantly influences the lambing number of the Tian Qiaoda red sheep through significance test analysis, and finally determining and screening SNP markers influencing the lambing number of the Tian Qiaoda red sheep. The genotypes determined by the method are GG, GA and AA, the individuals with the GG and GA genotypes are selected, the individuals with the AA genotype are eliminated, the breeding is improved, the lamb number of the Tian Qiaoda red sheep is increased, the multiple sheep variety is bred, and a scientific basis is provided for sustainable development of Xinjiang animal husbandry.
Drawings
FIG. 1 is a Manhattan diagram of a whole genome correlation analysis employing a generally linear model in a method of an embodiment of the invention;
FIG. 2 is a Manhattan diagram of a whole genome correlation analysis using a hybrid linear model in a method of an embodiment of the invention;
FIG. 3 is a graph of QQ-plot detection for two models in the method of an embodiment of the invention;
FIG. 4 is a gel electrophoresis chart of DNA detection of a red sheep neutralized Tian Qiaoda by the method of the present invention;
FIG. 5 is a diagram showing the typing structure of FSHR gene (Chr 11: g.75320741 site G > A) according to the method of the present invention.
Detailed Description
In order to further describe the technical means and effects adopted for achieving the preset aim of the present invention, the following detailed description of the specific implementation, technical scheme, features and effects according to the present invention will be given in the following preferred embodiments. The particular features, structures, or characteristics of the various embodiments in the description below may be combined in any suitable manner.
The technical terms involved in the embodiments of the present invention are explained as follows:
the SNP marker is derived from Tian Mianyang FSHR genes, follicle Stimulating Hormone Receptor (FSHR) of the SNP marker is a glycoprotein family member of a G protein coupled receptor superfamily, plays an important role in animal follicle development, FSHR is mainly expressed in follicular granulosa cells of superlambs and normal lambs, positive signals exist in original follicles, positive signals do not exist in the original follicles of normal adult sheep, and the expression quantity of FSHR is reduced in large dominant follicles.
KASP: the method is an abbreviation of competitive allele-specific PCR, and can accurately determine the double alleles of SNPs and lnDels at specific sites in a wide genomic DNA sample so as to genotype; the technology is based on specific matching of primer terminal base to genotype SNP and detect lnDels; in use, a Primer Mix composed of two allelic forward primers with different terminal bases and one reverse Primer is required to be prepared, and the 5' -ends of the two forward primers are respectively connected with detection Primer sequences with different sequences. The KASP high-throughput genotyping technique has higher throughput, faster speed, lower cost and more accurate results.
SLAF-seq sequencing technology: the specificity locus amplified fragment sequencing is a set of simplified genome sequencing technology, the SLAF technology has the characteristics of long effective reads, high flux, flexible scheme design and the like, represents a revolution in simplifying genome sequencing, brings about effective genome read length of 2x100bp, provides unprecedented enzyme digestion scheme customization service, develops up to 1 ten thousand tags at a time, acquires the most complete variation images (SNPs, lnDels) in the whole genome range, and enables researchers to select the polymorphism markers with the most information and reliability so as to realize the excellent capability of positioning important agronomic trait functional genes.
Example 1
Reagent and instrument equipment: a magnetic rack (Life Technologies DynaMagTM-2);
a centrifuge (eppendorf Centrifuge 5424); douglas Scientific NEXAR; douglas Scientific Soellex; douglas Scientific ARRAY; are all provided by the biological company of Baimeike;
mix: KASP 2 XMaster Mix KBS-1016-011 1536 FormulationV4.0 (supplied by LGC).
The specific experimental steps are as follows:
(1) Screening of sheep reproductive traits
Based on sheep reference genome, SLAF-seq sequencing technology is utilized, after PLink1.9 software quality control, full genome association analysis is carried out by utilizing a method of a general linear model and a method of a mixed linear model, as shown in figures 1-2, SNP loci with top1% are selected in the two methods, and differential SNP loci are screened out in the two methods as shown in figure 3; by gene annotation of the selected sites, FSHR genes were screened for genes that might affect lambing traits.
(2) Extraction of sheep genomic DNA
Collecting 5ml of the jugular vein blood of Tian Qiaoda red sheep by using a blood collection tube filled with sodium citrate anticoagulant, and preserving at-20 ℃; extracting genome DNA by using the kit, preserving at-20 ℃ for standby, and detecting the genome DNA, wherein the detection result of the genome DNA is shown in a gel electrophoresis chart shown in figure 4.
(3) Primer design and Synthesis
According to FSHR gene (GenBank ID: 443299) of sheep, primers were designed by using prime3 software with reference to SEQ ID NO.1, which is the nucleotide sequence of the SNP marker, and primers were synthesized by Shanghai Biotechnology Co., ltd.
The nucleotide sequence of the SNP marker is SEQ ID NO.1:
GGGTATGCTTGCCTCTTCTCCTTATAGAGATATTTCAAGAACCAGGATCC[G/A]TTCTCTGCCTAGCTATGGCTTAGAAAATCTTAAGAAGCTGCGGGCCAAGTCAACTT
the above [ G/A ] represents a mutant base.
The primer sequences were as follows: FSHR gene:
SEQ ID NO.2
upstream primer 1:5'-GAAGGTCGGATCAACGGATTGATATTTCAAGAACCAGGATCCA-3'
SEQ ID NO.3
Upstream primer 2:5'-GAAGGTGACCAAGTTCATGCTATATTTCAAGAACCAGGATCCG-3'
SEQ ID NO.4
Downstream primer 1:5'-CAGCTTCTTAAGATTTTCTAAGCC-3'
PCR amplification is shown in tables 1 and 2.
TABLE 1 PCR amplification reaction System
| Reactants | Volume of |
| 2×Master Mix | 2.5uL |
| Primer(s) | 0.07uL |
| DNA | 30-50ug |
| Water and its preparation method | Complement 5uL |
TABLE 2 PCR amplification reaction procedure
(4) KASP typing
SNP typing is carried out based on the specific matching of primer terminal base, and double allele judgment is carried out on SNPs by utilizing a Douglas platform of LGC company in England so as to carry out genotyping; the mutation of the chr11:g75320741 locus of the FSHR gene into 3 genotypes in the sheep group of the sheep and Tian Qiaoda is found; see fig. 5.
(5) Correlation analysis and application
3 genotypes were generated for the detected FSHR gene SNP sites using software SPSS 19.0: GG. The differences between the lambing numbers of AG, AA and Tian Qiaoda red sheep were tested for significance and the results are shown in Table 3.
TABLE 3 PCR amplification reaction procedure
Note that: mean±sd represents mean±standard deviation; the same row of data, with shoulders marked with different lowercase letters, indicates that the difference is significant (P < 0.05), and with shoulders marked with different uppercase letters, indicates that the difference is extremely significant (P < 0.01).
As can be seen from Table 3, the SNP locus of the FSHR gene is closely related to the number of lambing of Tian Qiaoda red sheep, the number of lambing of GG genotype is more than that of AA type, and the difference between the two is significant (P < 0.01).
In practical production application, individuals with genotypes GG and GA can be selected, individuals with genotype AA can be eliminated, the lambing number of Tian Qiaoda red sheep can be increased, and the cultivation of Xinjiang specialized multi-lamb variety can be accelerated.
The invention determines that the SNP molecular marker is derived from sheep FSHR gene by the method of the embodiment 1, and the specific position is Chr11, g.75320741 locus G > A (the mutation of the base G to the base A), wherein the number of the individual lambings of genotype GG is obviously higher than that of genotype AA; the SNP molecular marker can be used for identifying and screening sheep varieties with relatively large lambing numbers.
The invention uses the SNP marker to identify lambing number character, and simultaneously develops a primer for detecting the SNP marker, two forward primers (SEQ ID NO. 2-3) and one reverse primer (SEQ ID NO. 4):
upstream primer 1:5'-GAAGGTCGGAGTCAACGGATTGATATTTCAAGAACCAGGATCCA-3'
Upstream primer 2:5'-GAAGGTGACCAAGTTCATGCTATATTTCAAGAACCAGGATCCG-3'
Downstream primer 1:5'-CAGCTTCTTAAGATTTTCTAAGCC-3'
The invention can prepare a kit for detecting the lambing number character according to the SNP marker.
The SNP marker, the primer and the kit can be applied to the field of detecting the lambing number character or breeding sheep in the field of field.
The invention can adopt the SNP marker, the primer or the kit to detect the lambing character of Tian Mianyang, and the genotype GG individual is screened out from the lambing character to be used as a multi-yield variety, so that the lambing character can be applied to breeding of sheep in the field.
Although not exhaustive, those skilled in the art can choose from the prior art.
The foregoing disclosure is merely illustrative of specific embodiments of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art will readily appreciate variations or substitutions within the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Sequence listing
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Claims (5)
1. The FSHR gene specific SNP marker is characterized in that the SNP marker is derived from the FSHR gene of a red sheep and Tian Qiaoda, the SNP marker is positioned at 75320741 position on chromosome 3 of the red sheep and Tian Qiaoda, the original base is G, and the variant base is A; the nucleotide sequence of the SNP marker is SEQ ID NO.1.
2. A method of detecting and Tian Qiaoda red sheep lambing trait, the method comprising: determining the lambing number character of the red sheep of the sum Tian Qiaoda by detecting SNP markers in the gene of the red sheep of the sum Tian Qiaoda to be detected; wherein the SNP marker is the SNP marker according to claim 1.
3. A method of detecting and Tian Qiaoda a lambing trait in red sheep as claimed in claim 2, comprising the steps of:
extracting genome DNA of the sheep to be detected and Tian Qiaoda;
and (3) taking the DNA as an amplification template, taking sequences SEQ ID NO.2-4 as amplification primers, performing competitive allele-specific PCR typing, obtaining PCR typing results of GG type, AG type and AA type, determining all genotypes of SNP markers according to the typing results, and judging the lambing number characters of the to-be-detected and Tian Qiaoda red sheep according to the genotypes.
4. A method of detecting and Tian Qiaoda a lambing trait in red sheep as claimed in claim 3 wherein the PCR reaction conditions are: pre-denaturation at 95℃for 10min, denaturation at 95℃for 20s, circulation for 10 times, extension at 61-55℃for 1min, denaturation at 95℃for 20s, circulation for 26 times, and extension at 55℃for 1min;
the reaction system of the PCR:
2×Master Mix 2.5μL，
1.07. Mu.l of the upstream primer,
0.07. Mu.l of the upstream primer,
0.07. Mu.l of the downstream primer 1,
30-50 mu g of DNA template,
adding water to a total volume of 5 mu L;
the nucleotide sequence of the upstream primer 1 is SEQ ID NO.2;
the nucleotide sequence of the upstream primer 2 is SEQ ID NO.3;
the nucleotide sequence of the downstream primer 1 is SEQ ID NO.4.
5. The use of the SNP marker according to claim 1 as a detection target in breeding of red sheep of Tian Qiaoda, wherein individuals with genotype GG in the SNP marker are selected to breed red sheep breeds of Tian Qiaoda and with a large lambing number.
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